The MgcRacGAP SxIP motif tethers Centralspindlin to microtubule plus ends in Xenopus laevis.

Centralspindlin, a complex of the kinesin-6-family member MKLP1 and MgcRacGAP (also known as Kif23 and Racgap1, respectively), is required for cytokinesis and cell-cell junctions. During anaphase, Centralspindlin accumulates at overlapping central spindle microtubules and directs contractile ring formation by recruiting the GEF Ect2 to the cell equator to activate RhoA. We found that MgcRacGAP localized to the plus ends of equatorial astral microtubules during cytokinesis in Xenopus laevis embryos. How MgcRacGAP is stabilized at microtubule plus ends is unknown. We identified an SxIP motif in X. laevis MgcRacGAP that is conserved with other proteins that bind to EB1 (also known as Mapre1), a microtubule plus-end tracking protein. Mutation of the SxIP motif in MgcRacGAP resulted in loss of MgcRacGAP tracking with EB3 (also known as Mapre3) on growing microtubule plus ends, abnormal astral microtubule organization, redistribution of MgcRacGAP from the contractile ring to the polar cell cortex, and mislocalization of RhoA and its downstream targets, which together contributed to severe cytokinesis defects. Furthermore, mutation of the MgcRacGAP SxIP motif perturbed adherens junctions. We propose that the MgcRacGAP SxIP motif is functionally important both for its role in regulating adherens junction structure during interphase and for regulating Rho GTPase activity during cytokinesis.

MgcRacGAP restricts active RhoA at the cytokinetic furrow and both RhoA and Rac1 at cell-cell junctions in epithelial cells.

Localized activation of Rho GTPases is essential for multiple cellular functions, including cytokinesis and formation and maintenance of cell-cell junctions. Although MgcRacGAP (Mgc) is required for spatially confined RhoA-GTP at the equatorial cortex of dividing cells, both the target specificity of Mgc's GAP activity and the involvement of phosphorylation of Mgc at Ser-386 are controversial. In addition, Mgc's function at cell-cell junctions remains unclear. Here, using gastrula-stage Xenopus laevis embryos as a model system, we examine Mgc's role in regulating localized RhoA-GTP and Rac1-GTP in the intact vertebrate epithelium. We show that Mgc's GAP activity spatially restricts accumulation of both RhoA-GTP and Rac1-GTP in epithelial cells--RhoA at the cleavage furrow and RhoA and Rac1 at cell-cell junctions. Phosphorylation at Ser-386 does not switch the specificity of Mgc's GAP activity and is not required for successful cytokinesis. Furthermore, Mgc regulates adherens junction but not tight junction structure, and the ability to regulate adherens junctions is dependent on GAP activity and signaling via the RhoA pathway. Together these results indicate that Mgc's GAP activity down-regulates the active populations of RhoA and Rac1 at localized regions of epithelial cells and is necessary for successful cytokinesis and cell-cell junction structure.

Anillin regulates cell-cell junction integrity by organizing junctional accumulation of Rho-GTP and actomyosin.

Anillin is a scaffolding protein that organizes and stabilizes actomyosin contractile rings and was previously thought to function primarily in cytokinesis [1-10]. Using Xenopus laevis embryos as a model system to examine Anillin's role in the intact vertebrate epithelium, we find that a population of Anillin surprisingly localizes to epithelial cell-cell junctions throughout the cell cycle, whereas it was previously thought to be nuclear during interphase [5, 11]. Furthermore, we show that Anillin plays a critical role in regulating cell-cell junction integrity. Both tight junctions and adherens junctions are disrupted when Anillin is knocked down, leading to altered cell shape and increased intercellular spaces. Anillin interacts with Rho, F-actin, and myosin II [3, 8, 9], all of which regulate cell-cell junction structure and function. When Anillin is knocked down, active Rho (Rho-guanosine triphosphate [GTP]), F-actin, and myosin II are misregulated at junctions. Indeed, increased dynamic "flares" of Rho-GTP are observed at cell-cell junctions, whereas overall junctional F-actin and myosin II accumulation is reduced when Anillin is depleted. We propose that Anillin is required for proper Rho-GTP distribution at cell-cell junctions and for maintenance of a robust apical actomyosin belt, which is required for cell-cell junction integrity. These results reveal a novel role for Anillin in regulating epithelial cell-cell junctions.